Development of gene therapy for dystrophic epidemolysis bullosa with artificial adhension molecule

在营养不良性大疱性表皮松解症(EB)患者中，由于VII型胶原基因(COL7A1)的缺陷，对VII型胶原的免疫耐受必然被破坏。然而，由于许多EB患者表达在NC-1结构域之后截短的具有与基底膜结合的功能的异常的III型胶原，我们构建了由III型胶原的NC-1结构域和纤维连接蛋白胶原结合结构域嵌合体(NC-1/FNCBD)组成的人工分子的表达载体，以期在皮肤基底膜区域具有黏附活性和嵌合体分子的免疫耐受性。将NC-1/FNCBD表达载体导入HaCat角质形成细胞，可在细胞和培养上清液中高效表达嵌合体分子。为了将NC-1/FNCBD表达载体引入活体皮肤，必须开发新的技术将高分子分子，如质粒DNA，引入皮肤，因为皮肤的屏障功能足够强，可以阻止大于1 kDa的分子穿透。我们应用化学剥离和超声波能量将质粒DNA导入皮肤。50%羟基乙酸(GA)处理10min就足以打破角质层屏障功能，在GA处理的皮肤上施加US能量，可以将NC-1/FNCBD表达载体导入浸泡在质粒液中的皮肤中，导致NC-1/FNCBD分子在真皮-表皮交界处表达。

Because of type VII collagen gene (COL7A1) defect, immune tolerance for type VII collagen must be disrupted in the patients of dystrophic epidermolysis bullosa (EB). However, since many EB patients express abnormal type VII collagen truncated after NC-1 domain which function to bind to the basement membrane, we have constructed expression vector of the artificial molecule composed of NC-1 domain of type VII collagen and fibronectin collagen binding domain chimera (NC-1/FNCBD) to expect adhesive activity at cutaneous basement membrane region and immune tolerance of the chimera molecule. Transfection of the NC-1/FNCBD expression vector in HaCat keratinocytes provided efficient expression of the chimera molecule both in the cells and the culture medium. This artificial peptide may function to rescue severe blistering phenotype of the dystrophic EB patients.To introduce the NC-1/FNCBD expression vector to the living skin, novel technology must be developed to introduce high molecular weight molecule, such as plasmid DNA, to the skin, as barrier function of the skin is strong enough to prohibit penetration of the molecule bigger than 1 kDa in size. We applied chemical pealing and ultrasonic sound (US) energy to introduce plasmid DNA to the skin. 50% glycolic acid (GA) treatment for 10 minutes was enough to break horny layer barrier function, and application of US energy on the GA-treated skin enabled introduction of the NC-1/FNCBD expression vector to the skin soaked in the plasmid solution, resulting in the expression of the NC-1/FNCBD molecule at dermo-epidernal junction.